In a 2-year carcinogenesis bioassay, d-limonene (dL) induced kidney tumors in male F344 rats, but not in female F344 rats or either sex of mice. d-Limonene-1,2-oxide, a metabolite of dL, has been shown to bind reversibly to the male rat-specific urinary protein, α2u-globulin (α2u-G), resulting in an α2u-G-chemical complex that is more resistant to lysosomal degradation than α2u-G alone. This reduced degradation of α2u-G-chemical complex leads to an accumulation of this protein in the proximal convoluted tubules of the male rat kidney and to the morphological changes characteristic for α2u-globulin nephropathy. The only male rat strain known to be resistant to this renal disease is the α2u-G deficient NCI-Black-Reiter (NBR) rat. The objectives of this study were to determine whether or not dL causes sustained increases in cell proliferation and has promoting activity for renal adenomas in male rats and if the male rat-specific urinary protein, α2u-G, is required. In a 32-week initiation-promotion assay, male F344 and NBR rats were treated with either 0 or 500 ppm N-ethyl-N-hydroxyethylnitrosamine (EHEN) in the drinking water for 2 weeks. Experimental groups of 31 to 38 rats then received 0 or 150 mg d-limonene/kg/day in corn oil for 30 weeks by p.o. gavage 5 days/week. Cell proliferation in the proximal tubules was assessed via 5-bromo-2′-deoxyuridine-filled osmotic mini-pumps and immunohistochemistry after 7 weeks (2 weeks EHEN + 5 weeks dL) and at the end of the study (2 weeks EHEN + 30 weeks dL). Preneoplastic and neoplastic lesions were quantified in perfusion-fixed kidneys.
A 5-fold increase in the labeling index of P2-cells was found after 5 weeks and 30 weeks of promotion in all dL-treated F344 rats, whereas no difference between treatment groups was detected in NBR rats. No increase in tumors or preneoplastic lesions was detected in dL-treated NBR rats, whereas a 10-fold increase in renal adenomas and atypical hyperplasias was found in the EHEN-dL-treated F344 rats compared with F344 rats treated with EHEN-corn oil. d-Limonene treatment alone caused a significant increase in the number of atypical tubules and atypical hyperplasias in F344 rats when compared with the F344 vehicle control. On the other hand, a significantly lower incidence of liver tumors was found in EHEN-dL-treated F344 rats compared with F344 rats treated with EHEN-corn oil, suggesting a chemopreventative effect of dL on EHEN-induced liver carcinogenesis in F344 rats. It is thus concluded that dL promotes preneoplastic lesions and renal tumors only in the presence of the male rat-specific urinary protein, α2u-G. Since α2u-G is a species- and sex-specific protein that is causal for both the cytotoxic and carcinogenic response in the male rat, extrapolation of dL carcinogenicity data from rat studies to other species, including humans, is probably not warranted.